Proteins were the only known catalysts of cellular reactions until the discovery of RNA catalysts (ribozymes). In some instances, the folded structure of a ribozyme catalyses a cleavage reaction on another part of the same molecule (cis-reaction). In other instances (trans-reaction), the ribozyme may also act as a catalyst on another RNA or DNA molecule (substrate) by cleaving or ligating pieces of the substrate without changing itself in the process. (Zaug et al. Science, Vol. 231, pp. 470–75, 1986; Cech Science, Vol. 236, pp. 1532–39, 1987).
A well-characterized example of a ribozyme is the self-splicing Group I intron from the nuclear rRNA of Tetrahymena thermophila. An intron is an intervening sequence in a eukaryotic gene which does not encode a protein or in rare cases encodes a different protein. Introns are transcribed along with coding sequences (exons) to produce precursor RNA. The introns are removed from the precursor RNA and the exons are ligated by RNA cleaving and splicing steps. The Group I intron or ribozyme of T. thermophila catalyzes its own removal from the precursor RNA molecule. (Kruger et al. Cell 31:147–157, (1982); Zaug et al. (1986)). The self-splicing ribozyme catalyzes a variety of phosphodiester transfer reactions. The ribozyme can act as a ribonuclease, ligase, phosphotransferase, acid phosphatase, polymerase and RNA restriction endonuclease (Zaug, A. J., et al., Science 231:470–475 (1986); Zaug, A. J., et al., Nature 324:429–433 (1986); Zaug, A. J., et al., Biochemistry 25:4478–4482 (1986); Been, M. D., et al., Science 239:1412–1416 (1988); Doudna et al., Nature 339:519–522 (1989); all incorporated by reference herein).
The “hammerhead” and “hairpin” ribozymes also have been studied and described (Perreault et al., Nature 344:565–567 (1990); Perreault et al. Biochemistry 30:4020–25 (1991); Yang et al. Biochemistry 29:11156–60 (1990); Chowrira et al. Biochemistry 30:8518–22 (1991); Uhlenbeck Nature, 328:596–600 (1987)). The hammerhead ribozyme forms a stem loop secondary structure to form the catalytically active molecule. The hairpin ribozyme has a structure resembling a hairpin.
Although ribozymes are intriguing molecules, their use for in vivo applications is limited if not precluded. The all-RNA molecules are susceptible to degradation from enzymes (RNAses) present in vivo, There presently is no way known to inventors for delivering such molecules to the intended site in an active form.